The role of co-colonization in pneumococcal ecology and evolution

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Carina Valente

Dissertation presented to obtain the Ph.D degree in Biology | Molecular Biology

Instituto de Tecnologia Química e Biológica António Xavier | Universidade Nova de Lisboa

Oeiras,

December 2015

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First Edition, October 2015 Second Edition, January 2016 © Carina Valente

Cover image by Carina Valente ISBN: 978-989-206061

Financial support from Fundação para a Ciência e a Tecnologia,

Portugal through grant SFRH/BD/70058/2010 _{awarded to Carina}

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Dissertation presented on December 4, 2015, to obtain a Ph.D. degree in Biology | Molecular Biology by Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa.

Supervisors:

Raquel Sá-Leão Hermínia de Lencastre

Examiners:

Angela B. Brueggemann Krzysztof Trzciński

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Acknowledgments/Agradecimentos

The work presented in this thesis was only possible due to the support of several

people and institutions, that I would like to acknowledge here.

My PhD supervisor, Raquel Sá-Leão, for all the guidance and advice. Thank you for all

the opportunities you gave me and for helping me grow, both scientifically and as a

person. You once asked me if there was anyone that I admired scientifically: you are

on that list. You are my role model as a supervisor.

Professor Hermínia de Lencastre, co-supervisor of my thesis, for the opportunity of

doing this PhD and for all the advice along the years. For her demand of excellence,

which motivates us all to do our best.

ITQB, for the excellent working conditions and Fundação para a Ciência e a

Tecnologia for financial support.

The researchers and institutions with whom we have collaborated and that have

accepted me for internships during this PhD. Dr. Francisco Pinto at Faculdade de

Cièncias da Universidade de Lisboa; the late Professor Kathrin Mühlemann and Dr.

Sylvio Brugger at the Institute of Infectious Diseases, University of Bern; Dr. Jason

Hinds and Dr. Katherine Gould at the Bacterial Microarray Group, St. Georges

University of London; and Dr. Suzanne Dawid at the Department of Pediatrics and

Communicable Diseases, University of Michigan.

My Thesis Committee, Dr. Isabel Gordo and Professor Manuel Carmo Gomes, for their

critical appreciation of my work and for interesting discussions.

My former and current colleagues at the Microbiology of Human Pathogens Unit, for all

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My friends, for the joyful moments, for the support during the not-so-good moments

and for reminding me, when needed, that there is life outside the lab.

My family, my anchor. My idols and advisors in everything. The ones I reach for help

and to share good news. The ones who put up with my bad temper and are okay with

it. The ones I know I will always be able to count on, no matter what. This thesis is

dedicated to you. A minha família, minha âncora. Os meus ídolos e conselheiros em

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“[…] searching for the causes of things is the best way to spend a life.”

Aristotle

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Abstract

Streptococcus pneumoniae (or pneumococcus) is a frequent colonizer of the nasopharynx and an important cause of infectious diseases, with a high rate of

morbidity and mortality worldwide, particularly among young children, the elderly and

the immunocompromised. Despite this high burden of morbidity and mortality, invasive

pneumococcal disease is incidental. Nasopharyngeal colonization is the preferred

lifestyle for the pneumococcus, its prevalence being particularly high among young

children

Colonization by more than one S. pneumoniae strain, or co-colonization, is frequent and is important for pneumococcal biology because it promotes intra-specific

competition and evolution. Moreover, understanding co-colonization is critical to

monitor the impact of pneumococcal conjugate vaccines (PCV) and the extent of

serotype replacement by minor serotypes when vaccination targets the most abundant

ones. Despite its relevance and the fact that it has been recognized for several

decades, co-colonization remains poorly studied, mainly because high throughput

methods of multi-type detection have been developed only recently.

In this thesis we conducted five studies that aimed at understanding the dynamics of

pneumococcal co-colonization and how it is affected by pneumococcal conjugate

vaccines and by specific properties and mechanisms intrinsic to S. pneumoniae.

In the first study we used a molecular-based strategy to detect co-colonization in

nasopharyngeal samples from the pre- and vaccine eras, in order to evaluate the

impact of the introduction of the seven-valent pneumococcal conjugate vaccine (PCV7)

on pneumococcal colonization in Portuguese children. We showed that

co-colonization rates were significantly lower (p=0.004; Fisher’s exact test) among fully

vaccinated children (8.0%) than among children from the pre-PCV7 era (17.3%) or

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vaccinated children there was an asymmetric distribution of non-vaccine types (NVT)

found in single and co-colonization events and we have attributed this asymmetry to

differences in the competitive abilities of serotypes. We proposed that some NVTs

prevalent in the PCV7 era are more competitive than others, impairing their

co-existence in the same niche. The results obtained in this study may have important

implications since a decrease in co-colonization events is expected to translate in

decreased opportunities for horizontal gene transfer, hindering pneumococcal evolution

events, and might represent a novel potential benefit of conjugate vaccines.

The replacement of PCV7 by PCV13 (13-valent PCV) has raised the question on

whether this vaccine would have a similar effect on co-colonization. To address this

question, in the second study we used a similar approach to the one used in the study

conducted in the PCV7 era. We showed lower co-colonization prevalence in PCV13

fully vaccinated children, compared to children who had not received any PCV (20.6%

vs. 29.3%), an observation that supports the results obtained in the first study. As

before, we reported an asymmetric redistribution of NVTs in single and co-colonization

events in the vaccinated group, which supported our hypothesis of different competitive

abilities among NVT serotypes. Moreover, we showed that PCV13 serotypes are still

highly prevalent in a population with no universal but very high vaccine coverage,

mainly among non-vaccinated children. In vaccinated children PCV13 serotypes were

found mainly as minor serotypes, while in the non-vaccinated group these serotypes

were highly prevalent in single carriage events and were present in high relative

abundance in most co-colonization events. Overall, the results obtained in this study

regarding the impact of PCV13 on co-colonization corroborated the potential impact of

PCVs on pneumococcal ecology by decreasing co-colonization events and, thus,

decreasing opportunities for horizontal gene transfer.

In the third study we used knowledge previously obtained through the use of highly

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associated with the use of colony morphology for detection of co-colonization, a

convenient approach that has been the basis of most reports on co-colonization in

surveillance studies that do not systematically look for co-colonization. Comparison of

the serotype distribution of co-colonized samples detected through both methodologies

revealed that detection based on colony morphology resulted in a 20%

overrepresentation of serotypes that display very distinctive colony morphologies, such

as serotype 3 and non-encapsulated pneumococci, which did not reflect the real

epidemiology of these serotypes on the pneumococcal population.

In the fourth and fifth studies we explored how co-colonization might be affected by

specific properties and mechanisms intrinsic to S. pneumoniae biology.

In the fourth study we determined the impact of pherotype-mediated competition (or

competence stimulating peptide, CSP) on pneumococcal co-colonization using a

collection of co-colonized samples containing two strains of different serotypes. CSP

assignment of all pneumococci revealed that in 52.5% of the samples both strains were

of the same pherotype, while in 47.5% co-colonizing strains were of a different

pherotype. Comparison of the observed proportions of concordant and discordant

pherotypes with the ones estimated (53.8% and 46.2%, respectively), revealed no

significant differences. The results obtained in this study supported the hypothesis that

there is a limited role of pherotype-mediated competition on co-colonization.

In the fifth study we explored the impact of the blp (bacteriocin-like peptide) locus and

bacteriocin secretion on co-colonization. We characterized the blp locus of a highly diverse collection of co-colonizing strains and determined their inhibitory activity

through competition overlay assays. For comparison, we performed the same

characterization in a collection of pneumococci isolated from single carriage events.

We showed that co-colonizing pneumococci present high genetic diversity at the level

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showed also that pneumococcal strains co-colonize individuals independently of the

genetic content of their blp-locus and independently of their phenotypes of bacteriocin

secretion, and that phenotypes of bacteriocin secretion are the same in strains isolated

from single and co-colonization events. Overall, the results obtained in this study

support the hypothesis that there is a limited role of the blp-locus and bacteriocin secretion on co-colonization.

In conclusion, the results obtained in this thesis improved the scarce knowledge on

pneumococcal co-colonization through the use of innovative methodologies and by

addressing important questions such as the impact of PCVs and bacterial competition

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Resumo

A bactéria Streptococcus pneumoniae (pneumococo) coloniza assintomatica e frequentemente a nasofaringe e é também uma importante causa de doença

infecciosa, com elevadas taxas de mortalidade e morbilidade, sobretudo em crianças,

idosos e indivíduos imuno-comprometidos. A colonização da nasofaringe é, no

entanto, preferencial no ciclo de vida do pneumococo, sendo particularmente elevada

em crianças até aos seis anos.

A colonização da nasofaringe por mais do que uma estirpe de S. pneumoniae, ou

co-colonização, é frequente. O estudo da co-colonização é importante dada a sua

relevância para a biologia do pneumococo, por promover interacções intra-específicas

e, consequentemente, a evolução da espécie. Além disso, compreender a

co-colonização é fundamental para a monitorização do impacto das vacinas

pneumocócicas conjugadas (PCV) e do grau de substituição de serótipos por serótipos

minoritários, num cenário em que a vacina é dirigida contra os serótipos mais

abundantes. Apesar de a co-colonização ser reconhecidamente importante e um

fenómeno identificado há já várias décadas, continua pouco estudada, sobretudo

devido ao facto de apenas recentemente terem sido desenvolvidos métodos

adequados à sua detecção.

No âmbito desta tese foram realizados cinco estudos com o objectivo de entender a

dinâmica da co-colonização por pneumococos e qual o impacto das PCVs e de certas

propriedades e mecanismos intrínsecos à biologia desta bactéria neste fenómeno.

Com o objectivo de avaliar o impacto da introdução da vacina pneumocócica

conjugada sete-valente (PCV7) na co-colonização por pneumococos em crianças

portuguesas, realizámos o primeiro estudo, recorrendo a métodos moleculares para

detectar co-colonização em amostras da nasofaringe de crianças obtidas antes e após

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completa apresentavam uma prevalência de co-colonização significativamente inferior

(8.0%) à de crianças não vacinadas no mesmo período (18.3%) ou no período

pré-vacinal (17.3%). Nas crianças vacinadas observou-se uma distribuição assimétrica dos

serótipos não vacinais (NVT) nos eventos de co-colonização e colonização simples

(i.e., colonização por uma só estirpe), assimetria essa que atribuímos a diferenças nas

capacidades competitivas dos serótipos. Esta observação levou à proposta de que

alguns NVT prevalentes na era vacinal são muito competitivos, impedindo a sua

co-existência com outros serótipos no mesmo nicho. Os resultados deste estudo poderão

ter implicações relevantes dado que uma diminuição nos eventos de co-colonização

poderá traduzir-se numa diminuição das oportunidades para a ocorrência de

transferência horizontal de genes e, consequentemente, dos eventos de evolução,

representando potencialmente um novo benefício das vacinas conjugadas.

A substituição da PCV7 pela PCV13 (PCV treze-valente) levou-nos a questionar se

esta vacina teria um efeito semelhante ao da PCV7 na co-colonização. Deste modo,

no segundo estudo adoptámos uma abordagem semelhante à do estudo da era da

PCV7. Demonstrámos que a prevalência de co-colonização em crianças com

vacinação completa (20.6%) era inferior à de crianças não vacinadas do mesmo

período (29.3%). Em concordância com o estudo anterior, observámos uma

distribuição assimétrica de NVT em eventos de co-colonização e colonização simples,

facto que suporta a hipótese previamente proposta de diferenças nas capacidades

competitivas dos serótipos. Demonstrámos ainda que os serótipos incluídos na PCV13

são ainda prevalentes na população. Nas crianças vacinadas os serótipos incluídos na

PCV13 foram encontrados como um serótipo minoritário na maioria das ocorrências,

enquanto que no grupo de não-vacinados estes serótipos eram muito prevalentes em

eventos de colonização simples ou como serótipo maioritário em eventos de

co-colonização. Os resultados apresentados neste estudo relativos ao impacto da PCV13

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pneumococo pela diminuição dos eventos de co-colonização e das oportunidades para

transferência horizontal de genes.

No terceiro estudo usámos informação obtida previamente através do uso de métodos

moleculares na detecção de co-colonização para quantificar o viés associado à

detecção de co-colonização com base na morfologia das colónias de pneumococos.

Dada a sua conveniência, esta abordagem baseada na morfologia constitui a base da

maioria dos dados reportados na literatura relativos à prevalência de co-colonização. A

comparação da distribuição de serótipos em amostras co-colonizadas com detecção

efectuada através de ambas as metodologias demonstrou que a detecção com base

na morfologia das colónias aumenta em 20% a prevalência de serótipos que

apresentam morfologias mais facilmente distinguíveis, como é o caso do serótipo 3 e

dos pneumococos não-encapsulados, podendo alterar artificialmente a prevalência

estimada destes serótipos na população.

No quarto e quinto estudos, explorámos o impacto de propriedades e mecanismos

intrínsecos à biologia do pneumococo na co-colonização.

No quarto estudo avaliámos o impacto da competição mediada pelo ferótipo (ou

péptido indutor da competência) na co-colonização, recorrendo a uma colecção de

amostras co-colonizadas por duas estirpes de pneumococos. A determinação do

ferótipo para todas as estirpes revelou que 52.5% das amostras continham estirpes

com o mesmo ferótipo, enquanto que nas restantes as estirpes eram de ferótipos

diferentes. A comparação das proporções de amostras com ferótipos concordantes

(52.5%) e discordantes (47.5%) com as proporções estimadas (53.8% e 46.2%,

respectivamente) não revelou diferenças significativas. Estes resultados sugerem que

a competição mediada pelo ferótipo tem um impacto limitado na co-colonização.

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na co-colonização. Caracterizámos o locus de uma colecção muito diversa de

pneumococos identificados em eventos de co-colonização e determinámos a sua

capacidade inibitória através de ensaios de competição em meio sólido. Utilizámos

como controlo uma colecção de pneumococos isolados de eventos de colonização

simples. Demonstrámos que os pneumococos a co-colonizar apresentam uma elevada

diversidade genética ao nível do locus blp e exibem diferentes fenótipos de secreção

de bacteriocinas e de imunidade. Adicionalmente, reportámos que os pneumococos

co-colonizam um hospedeiro independentemente dos fenótipos de secreção de

bacteriocinas e que os mesmos não são diferentes em estirpes de co-colonização e de

colonização simples. Os resultados obtidos neste estudo sugerem que o impacto do

locus blp e da secreção de bacteriocinas na co-colonização é limitado.

Em suma, os resultados apresentados nesta tese aumentaram o conhecimento sobre

a co-colonização por pneumococos através do uso de metodologias inovadoras e da

resposta a questões importantes, tais como o impacto das PCV e dos mecanismos de

competição neste fenómeno, e a forma como os mesmos podem influenciar a ecologia

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Thesis outline

The purpose of this thesis was to gain insights on the dynamics of pneumococcal

co-colonization and how it is affected by pneumococcal conjugate vaccines and by

specific properties and mechanisms intrinsic to S. pneumoniae biology.

Chapter I is a general introduction where important aspects of S. pneumoniae epidemiology and biology, relevant for the scope of the thesis, are presented. Among

these, are pneumococcal epidemiology, effects of anti-pneumococcal vaccination,

importance of co-colonization, methods for detection of co-colonization, and bacterial

properties and competition mechanisms that might impact on co-colonization.

Chapter II describes the assessment of co-colonization in a collection of

nasopharyngeal samples obtained from vaccinated and non-vaccinated children,

encompassing the availability of PCV7 in Portugal, aiming at determining the impact of

this vaccine on co-colonization. The analysis includes the combination of two sensitive

molecular methods for detection of co-colonization and comparison of co-colonization

patterns among children sampled in the pre-PCV7 era, and vaccinated and

non-vaccinated children sampled in the PCV7 era.

Chapter III describes a study conducted to evaluate the impact of PCV13 on

co-colonization. Samples obtained from PCV13 vaccinated and non-vaccinated children

were analyzed with a capsular microarray to detect co-colonization and the two groups

were compared regarding co-colonization patterns.

Chapter IV describes a study conducted to assess the bias in the serotype distribution

of pneumococci associated with co-colonization detection based on colony

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distribution of co-colonized samples detected by molecular methods and by colony

morphology.

Chapter V describes the impact of pherotype-mediated fratricide on co-colonization.

The analysis included pherotype assignment of a co-colonized collection of

nasopharyngeal samples and estimation of the proportion of samples co-colonized with

strains of the same or of a different pherotype for comparison with the observed

proportions.

Chapter VI describes the impact of the blp (bacteriocin-like peptide) locus and bacteriocin secretion on pneumococcal co-colonization. The analyses included genetic

characterization of the blp locus of co-colonizing strains and assessment of the phenotype of bacteriocin secretion through overlay assays. Comparison with a control

collection of strains isolated from single carriage events was also performed.

Chapter VII presents general conclusions of the studies conducted in this thesis and

enumerates several questions that remain unanswered and could be the focus of future

research.

Chapters II, IV and V are reproductions of the following publications and can be read

independently:

Chapter II - Valente, C., J. Hinds, F. Pinto, S. D. Brugger, K. A. Gould, K.

Mühlemann, H. de Lencastre, R. Sá-Leão. 2012. Decrease in pneumococcal

co-colonization following vaccination with the seven-valent pneumococcal conjugate

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Chapter IV - Valente, C., H. de Lencastre, R. Sá-Leão. 2013. Selection of distinctive

colony morphologies for detection of multiple carriage of Streptococcus pneumoniae. Pediatr. Infect. Dis. J.; 32(6):703-4. doi: 10.1097/INF.0b013e31828692be.

Chapter V - Valente, C., H. de Lencastre, R. Sá-Leão. 2012. Pherotypes of

co-colonizing pneumococci among Portuguese children. Microbe Drug Resist;

18(6):550-4. doi: 10.1089/mdr.2011.0228.

Chapter III has been submitted for publication and Chapter VI is nearly ready for

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Chapter I

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Streptococcus pneumoniae, on the vanguard of scientific

discoveries

Streptococcus pneumoniae, or pneumococcus, is an important human pathogen, continuing to kill thousands of people and being still “the captain of the men of death”,

as William Osler called it in 1918 (Osler, 1901).

The first reports about “elongated diplococci” in infected lungs are from Edwin Klebs

and date back to 1875. It was only in 1881, however, that this bacterium was first

isolated and its pathogenic potential was established, by both Louis Pasteur and

George Sternberg (reviewed in (Watson et al., 1993)).

Several designations were attributed to the pneumococcus along the years (reviewed

in (Watson et al., 1993)) taking into consideration the shape and type of disease

caused by the bacteria but, in 1974, the name Streptococcus pneumoniae was adopted

based on its morphology in chains when grown in liquid medium (Bergey, 1974).

Due to its important role as a human pathogen, S. pneumoniae is one of the most well

studied bacteria. For this reason, it is not surprising that it has been center stage to

important medical and scientific discoveries. In the field of medicine, the

pneumococcus was used in the development of Gram’s stain that allowed the

distinction between Gram positive and Gram negative pathogenic bacteria (reviewed in

(Watson et al., 1993)), the discovery of the ability of polysaccharides to induce antibody

production and their use as antigens in vaccines (Avery, 1917; Heidelberger & Avery,

1923), and the confirmation of the therapeutic efficacy of penicillin (Tillett et al., 1944).

In science, S. pneumoniae was the leading character in the discovery of the “Transforming Principle” and the mechanism of bacterial gene transfer (Griffith, 1928),

in the identification of DNA as the genetic material (Avery et al., 1944), in the discovery

of the protective role of the capsule against host phagocytic cells (Felton et al., 1955),

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all of which have contributed to even greater discoveries and, as of today, are still

being used in the most various fields and applications.

Over 130 years have passed since the discovery of the pneumococcus and science

and medicine are still far from winning the battle against this highly successful

pathogen. Recent estimates on the number of serious cases of pneumococcal disease

reach over 14 million, from which over 0.8 million cases result in the death of children

below five years of age (O'Brien et al., 2009) .

Streptococcus pneumoniae

S. pneumoniae is a Gram positive bacterium with the form of an elongated coccus (lancet-shaped). It varies between 0.5 and 1.5 µm in diameter and can be found

isolated in single cells, in the form of diplococci, and in longer chains. Pneumococci are

facultative anaerobic, display α-hemolytic activity when grown on blood-supplemented

plates and are typically optochin susceptible and soluble in bile salts. It is a fastidious

organism, its growth being favored in media containing blood, at 37ºC, and in a

CO2-supplemented atmosphere (CDC, 2012; Sneath, 1986).

The genome of S. pneumoniae is a covalently closed circular DNA molecule of ~2M base pairs, varying, in size and content, from strain to strain. It contains over 1500

genes that are essential for cell viability and a variable number of genes essential for

virulence or to maintain a non-invasive phenotype (Bijlsma et al., 2007; Hava & Camilli,

2002; Lanie et al., 2007; Obert et al., 2006; Orihuela et al., 2004). S. pneumoniae possesses a highly plastic genome, with several recombination hotspots, which

constitutes an evolutive advantage and makes it one of the most well adapted

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2007; Obert et al., 2006). In fact, analysis of 17 sequenced genomes showed that only

46% of the homologous gene clusters were common between strains (Hiller et al.,

2007).

Due to all the diversity in pneumococcal population and to the need to classify and

cluster pneumococcal strains in the context of epidemiological studies, a gold-standard

fingerprinting method, MLST (multi-locus sequence typing) is generally used as a

typing method, in which a sequence type (ST) is assigned to a strain based on the

combination of the allelic variants of seven housekeeping genes – aroE, gdh, gki, recP,

spi, xpt, and ddl (Enright & Spratt, 1998) (http://www.mlst.net/).

One of most distinctive characteristics of the pneumococcus is its polysaccharide

capsule, which led Avery to name it “the sugar-coated microbe” (Bardossi, 1988). This

capsule has antigenic properties which can be used as an identification method,

through the Quellung reaction, by the use of specific antisera (Sorensen, 1993). The

capsule is the main pneumococcal virulence factor and has several distinct functions

that will be discussed later in this chapter. Based on different antigenic properties

conferred by biochemical and structural differences of the polysaccharide capsule, over

95 capsular types, or serotypes, have been described up to now (Calix & Nahm, 2010;

Jin et al., 2009; Oliver et al., 2013; Park et al., 2007; Park et al., 2015).

Epidemiology of S. pneumoniae

Pneumococcal colonization

S. pneumoniae is a frequent inhabitant of the human nasopharynx, co-existing commensally with the human host. Colonization can occur soon after birth and remains

high in the first three years of life, decreasing until the age of ten and remaining low

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pneumococcus at least once in life and each serotype can colonize for several weeks

or months, being then replaced by another serotype or reacquired (Gray et al., 1980;

Sá-Leão et al., 2008).

Carriage is usually asymptomatic and its duration varies according to the serotype of

the strain, the age of the child and the immunological status, as it allows the individual

to acquire B cell mediated immunity against the carried serotype. A low mucosal

immunity will result in persistent and repeated colonization events, while a stronger

local response will accelerate clearance and prevent re-colonization (Bogaert et al.,

2004a; Weinberger et al., 2008).

Asymptomatic carriers constitute a reservoir of pneumococci in the community and are

important vehicles of transmission, which can occur via direct contact between

individuals, by aerosols, or by contact with contaminated abiotic surfaces (Musher,

2003; Walsh & Camilli, 2011).

Risk factors for pneumococcal carriage include young age (up to 2 years of age),

regular contact with young children, crowding (as occurs in day care centers, prisons,

hospitals, and military camps), previous respiratory disease, both infectious and

chronic, and cigarette smoking (Abdullahi et al., 2012; Almeida et al., 2014; Bogaert et

al., 2004a; Gray et al., 1980; Labout et al., 2008).

Several studies have been conducted worldwide on the subject of pneumococcal

carriage (Adegbola et al., 2014; Cohen et al., 2012; Daana et al., 2015; Vestrheim et

al., 2010) (Nunes et al., submitted), most of them focused on young children attending day care centers, as this is the age group where colonization is more frequent and

crowding is high in day care centers, favoring transmission (Bogaert et al., 2004a;

Sá-Leão et al., 2008). These studies have shown that the carriage rates and the

distribution of the carried serotypes vary across geographical locations, reaching

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(Vestrheim et al., 2010). In Portugal, studies conducted in the Lisbon area report a

pneumococcal carriage rate of ~64% in 2010 (Nunes et al., submitted).

Pneumococcal disease

From the nasopharynx pneumococci can spread to other body sites to cause a wide

range of diseases that vary in prevalence and severity. The most common forms of

disease are mild infections such as acute otitis media (AOM) and sinusitis. The

nasopharynx is connected to the middle ear via the Eustachian tube which, if clogged,

can trap bacteria and cause a middle ear infection. Despite being a mild infection, AOM

has a high socio-economic impact as it is the leading bacterial infection in children in

high income countries and the primary reason for antibiotic prescription (Hau et al.,

2013). In fact, by the age of 3 years, over 80% of the children are estimated to have an

AOM episode and over 40% to have recurrent episodes (Vergison et al., 2010).

Overall, the serotypes more frequently associated with AOM are serotype 3, 6A, 6B,

9V, 11A, 14, 19A, 19F and 23F, although this may vary with geographical location and

vaccine coverage (discussed below in this chapter) (Rodgers et al., 2009).

The pneumococcus can also be responsible for more severe infections, such as

pneumonia (non-invasive disease), bacteremia and meningitis (invasive disease).

Pneumonia is the non-invasive pneumococcal disease with highest burden in adults. It

has high incidence rates and high mortality risk, especially in the elderly, and is the

most common infectious source for invasive pneumococcal disease (Drijkoningen &

Rohde, 2013).

The incidence of invasive pneumococcal disease (IPD) in any population depends on

several factors, such as geographic location, season of the year, serotype prevalence,

age, co-morbidities, and vaccine coverage (Drijkoningen & Rohde, 2013; Hausdorff et

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age, children younger than 2 years of age, and those with certain underlying conditions

that compromise the immune system, such as HIV infection and chronic diseases.

The World Health Organization estimated the occurrence of 14.5 million global cases of

serious illness in children less than 5 years old (O'Brien et al., 2009). According to the

USA Active Bacterial Core surveillance (ABCs) database of the Emerging Infections

Program Network, in 2013, the incidence of invasive pneumococcal disease in

individuals >65 years of age was 30.5 cases per 100,000 population and, in infants <1

year, the incidence was 29.8 cases per 100,000 population (CDC).

The serotype prevalence in disease also varies with age, vaccine coverage and type of

disease. Serotypes 4, 6B, 9V, 14, 18C, 19F and 23F were the most prevalent in

pediatric invasive disease in the USA before the introduction of the seven-valent

pneumococcal conjugate vaccine (PCV7) (Hausdorff et al., 2005), while serotype 19A

emerged after PCV7 introduction (Richter et al., 2009). Serotypes 1, 4 and 14 have

been associated with bacteremia (Hausdorff et al., 2000). Serotype 1 has also been

associated with pneumonia, together with serotype 3 (Hausdorff et al., 2005).

Figure 1. Incidence rate of invasive pneumococcal disease in children under 5 years

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In Portugal, a study has shown that serotypes 1, 7F and 19A accounted for 61% of

pediatric invasive pneumococcal disease cases between 2006 and 2008 (Aguiar et al.,

2010).

From the information provided above it is clear that, although there are over 95

serotypes described, only a few are responsible for causing disease. Several studies

have aimed at establishing differences in invasive potential among serotypes

(Brueggemann et al., 2003; Brueggemann et al., 2004; Greenberg et al.; Sá-Leão et

al., 2011; Sleeman et al., 2006). These studies had different designs and were conducted in different geographical locations but the overall results were similar, as

most reported a high invasive disease potential for serotypes 1, 3, 4, 5, 7F, 9V, 14, and

18C. The study conducted in Portugal identified the same serotypes as having a high

invasive potential, among others, while serotypes 6A, 6B, 11A, 15B/C, 16F, 19F, and

23F, among others, to be mostly associated with colonization (Sá-Leão et al., 2011).

More importantly, this study has shown that different genetic lineages of the same

serotype have different invasive potential, as is the case of serotypes 3, 6A, 6B, 19A,

19F, and 23F, among others.

Non-susceptibility to antimicrobial agents

Non-susceptibility of S. pneumoniae to commonly used antibiotics has been reported worldwide (CDC; ECDC, 2013) and has increased dramatically since the description of

the first clinical isolate resistant to an antimicrobial agent (penicillin) in 1967 (Hansman

et al., 1971).

The distribution of non-susceptible isolates seems to be associated with the capsular

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pneumococcal conjugate vaccine coverage (Dagan & Klugman, 2008; Goossens,

2009; Hausdorff et al., 2005; Riedel et al., 2007).

In general, high rates of non-susceptibility are observed in countries with high antibiotic

consumption, such as Southern and Eastern European countries, whilst low

non-susceptibility rates are observed in countries with low antibiotic consumption, such as

Northern European countries (Goossens, 2009; Riedel et al., 2007). Accordingly,

strains isolated from carriage tend to be more frequently non-susceptible to

antimicrobial agents than strains isolated from disease episodes (Hausdorff et al.,

2005).

The latest report from the European Center for Disease Control and Prevention

(ECDC) showed that, in 2013, the percentage of isolates non-susceptible to penicillin

causing invasive disease ranged between < 1.1% (the Netherlands) and 40.0%

(Cyprus), while for macrolides non-susceptibility rates ranged between 1.5% (Latvia)

and 38.1% (Romania) (ECDC, 2013). In the USA, in the same year, the percentages of

penicillin and macrolide non-susceptible isolates were 4.7% and 28.5%, respectively

(CDC).

In Portugal, data from carriage isolates from 2010 showed percentages of

non-susceptibility to penicillin and macrolides of 19.6% and 25.2%, respectively. The same

study showed that in that period, non-susceptibility to those antimicrobial agents was

associated mainly with serotypes 6C, 15A, 19A, 19F, and non-encapsulated isolates

(Nunes et al., submitted).

A study focused on isolates causing invasive disease in Portugal, from 2006-2008,

showed that the rates of non-susceptibility to penicillin and macrolides were 18.7% and

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antimicrobial agents was associated mainly with serotypes 19A, 14, 19F, 6C and 23F

(Aguiar et al., 2010).

Anti-pneumococcal vaccination

Being S. pneumoniae “the captain of the men of death” (Osler, 1901), it is not surprising that several attempts have been made throughout history to prevent

pneumococcal disease through vaccination.

Although not effective, the first attempt was the use of killed whole bacterial cells to

immunize mineworkers, in 1911, to prevent death by pneumonia (Wright et al., 1914).

The demonstration of the efficacy of pneumococcal vaccines was a long process,

mainly due to flawed study designs and serious adverse effects of the vaccines.

Although some clinical trials were undertaken, the discovery of antibiotics and their

efficacy against pneumococcal disease put this process on hold, until Robert Austrian

proved the efficacy of pneumococcal polysaccharide vaccines and, as a consequence,

the 14-valent polysaccharide vaccine was licensed (Austrian et al., 1976). In 1983 this

vaccine was extended to 23 serotypes and licensed, being still in the market with the

commercial name of Pneumovax© 23 (PPV23). PPV23 is immunogenic for serotypes

1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19F, 19A, 20, 22F,

23F, and 33F when administered in adults, the elderly and children older than 2 years

of age, as in younger children, who have an underdeveloped immune system, it fails to

induce an adequate immune response (Bogaert et al., 2004b; Douglas et al., 1983).

In 2000, the first pneumococcal conjugate vaccine (PCV7) was licensed and introduced

in the National Immunization Plan in the USA, with the commercial name of Prevnar©,

and protecting against the seven serotypes more prevalent in pneumococcal disease in

children under 6 years of age: 4, 6B, 9V, 14, 18C, 19F, and 23F (CDC, 2000). The

polysaccharide of these serotypes was conjugated to a protein carrier, CRM197,

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Two additional conjugate vaccines have been developed in more recent years: a 10-

and 13-valent vaccine, in 2009 and 2010, respectively (Synflorix© and Prevnar©13,

respectively). Synflorix© (PCV10) includes the 7 serotypes present in PCV7 and 3

additional serotypes - 1, 5 and 7F – conjugated to Haemophilus influenza protein D as

the carrier protein. Prevenar©13 (PCV13) replaced PCV7 in 2010 and includes six

additional serotypes, conjugated to the same carrier protein, CRM197: 1, 3, 5, 6A, 7F,

and 19A.

In Portugal, PCV13 was introduced in the National Immunization Plan for children born

after January 2015 on a scheme of two doses followed by a booster dose.

Nevertheless, PCVs have been available in the market since June 2001 (PCV7), April

2009 (PCV10) and January 2010 (PCV13) and their use in young children has been

recommended by the Pediatric Portuguese Society. Estimates on PCV7 coverage in

2009 reached 62%, while PCV10 coverage was estimated to be 13% in the same year

(data from the National Statistics Institute (INE) and IMS).

Effects of pneumococcal vaccination

The introduction of PCV7 has led to several changes in pneumococcal epidemiology

and population structure. These changes are more or less significant according to the

vaccine coverage and geographic location and included: (i) decrease in IPD in

vaccinated children, (ii) decrease in IPD in non-vaccinated children and in adults, as a

reflex of the herd-immunity effect, (iii) increase in the incidence of IPD caused by

non-vaccine serotypes, and (iv) replacement of non-vaccine types by non-non-vaccine types in

carriage. The USA, a country with immediate massive PCV7 use, is a good example of

all these changes. A 77% decrease in IPD cases in vaccinated children was reported

four years after vaccine introduction (CDC, 2008) and later a 45% decrease in IPD in

all age groups was observed, accompanied by a 94% reduction of vaccine types

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to non-vaccine types also increased in the first years of vaccination, particularly due to

serotype 19A (Hicks et al., 2007).

In Europe, the same effects were observed but with some variability among countries,

depending on vaccine coverage. Decrease in IPD incidences varied between 28% and

68% and serotype replacement was also observed, with non-vaccine serotypes 1, 19A,

3, 6A, and 7F being the ones with higher incidence in IPD after widespread use of

PCV7 (reviewed in (Isaacman et al., 2010)).

In Portugal, the same scenario was observed. The incidence of IPD cases associated

with PCV7 serotypes was reduced from 56% (before PCV7 introduction) to 17% by

2006-2008, in children aged up to 17 years of age (Aguiar et al., 2010). Carriage of

PCV7 serotypes also decreased, while the prevalence of non-PCV7 serotypes

increased, maintaining the overall prevalence of carriage in the population (Sá-Leão et

al., 2009) (Nunes et al., submitted).

Since serotypes included in PCV7 are frequently resistant to antimicrobial agents,

introduction of PCV7 vaccination was predicted to have an impact on the levels of

antimicrobial resistance worldwide (Dagan & Klugman, 2008; Kyaw et al., 2006). This

decrease was, in fact, observed following the first years of vaccination but was rapidly

compensated by the emergence of drug-resistant non-PCV7 serotypes (Mera et al.,

2009). In Portugal, antimicrobial resistance rates were maintained, despite the

widespread use of PCV7 (Simões et al., 2011) (Nunes et al., submitted).

PCV10 and PCV13 have similar effects on the population as the ones described for

PCV7, with reports on the impact these vaccines becoming more frequent (Hammitt et

al., 2014; Jokinen et al., 2015; Moore et al., 2015; Waight et al., 2015). In the USA a study reported a decrease of 93% in IPD cases attributable to PCV13-only serotypes,

in children under 5 years old, and a decrease of 58-72% in adults, depending on age,

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serotypes IPD cases was of 69% for all ages (Waight et al., 2015). However, both

studies reported an increase in IPD cases associated with non-PCV13 serotypes,

particularly in children under 5 years old, evidence of the serotype replacement

expected to occur.

Despite the serotype replacement that has been observed and that is attributable to the

fact that current vaccines only target a limited number of serotypes, the strategy of

targeting the most prevalent serotypes in IPD has been highly successful. However,

the adaptability of the pneumococcal population to these human interventions has been

a driving force for the development of the ideal vaccine. This vaccine would be a

conserved, universally present surface protein, capable of eliciting a good immune

response in all age groups. No such protein has been described so far but several

initiatives are ongoing, using surface proteins, combinations of proteins or an whole cell

approach to find a vaccine that could ultimately overcome the limitations of currently

used conjugate vaccines (reviewed in (Feldman & Anderson, 2014)).

Pneumococcal co-colonization

Biological significance

The preferential niche of the pneumococcus is the nasopharynx, a polymicrobial

environment where the pneumococcus is forced to interact with other microorganisms

of the same or different species. Simultaneous colonization by more than one

pneumococcal strain, or co-colonization, is frequent and is of high clinical,

epidemiological and ecological relevance (O'Brien & Nohynek, 2003; Shak et al.,

2013).

It has been shown that colonizing pneumococcal strains can interfere both negatively

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2000; Marks et al., 2012b). Marks et al. (Marks et al., 2012b) have shown in a mouse model that strains with poor colonization efficiency have their efficiency improved when

in co-colonization with a highly efficient strain that is already established. This

improvement was independent of genetic exchange between strains. On the other

hand, Lipsitch et al. (Lipsitch et al., 2000) have shown, also in a mouse model, that carriage of an established strain can inhibit acquisition of a second strain in a strain-

and density-dependent manner. The inhibition was observed through a reduced

colonization probability and/or through reduced cell counts in the colonized animals.

Additionally, some molecular mechanisms have been shown to contribute to inter-strain

interactions, namely bacteriocin production and competence-mediated fratricide, which

will be discussed later in this chapter (Dawid et al., 2007; Guiral et al., 2005).

Co-colonization is a requirement for horizontal gene transfer between pneumococci

through homologous recombination, a mechanism important for evolution in this

species (Coffey et al., 1991; Feil et al., 2000; Smith et al., 1993; Spratt et al., 2001).

Several studies reported the occurrence of genetic exchange in biofilm models (Carrolo

et al., 2014; Marks et al., 2012b; Wei & Havarstein, 2012) and also in in vivo models (Marks et al., 2012b). Additionally, Hiller et al. (Hiller et al., 2010) have shown the occurrence of genetic rearrangements in vivo that generated genetic diversity during a

pediatric chronic infection. Several population genetic studies have also shown the

occurrence of genetic recombination, including capsule switching events (Croucher et

al., 2011; Donkor et al., 2011). Donkor et al. (Donkor et al., 2011) have used an MLST approach to characterize pediatric colonization strains and they found evidence of

extensive recombination among those strains, which they have related to the high

prevalence of co-colonization in the population.

Besides the evolutionary implications, DNA exchange between co-colonizing strains

can have important clinical and epidemiological consequences, not only because it has

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significant changes in the virulence of a strain (Hiller et al., 2011), but also because this

genetic exchange has played a role in the development and evolution of antibiotic

resistance (Dowson et al., 1989). The occurrence of capsule switching events is also of

importance as it can have an impact on the virulence of strains and, in the context

vaccine effectiveness, can result in the emergence of vaccine escape recombinants

(Brueggemann et al., 2007). In the same context, understanding co-colonization is

critical to monitor the extent of serotype replacement by minor serotypes when

vaccination targets the most abundant ones (Lipsitch, 1999; Lipsitch, 2001; Spratt &

Greenwood, 2000).

Another important clinical implication is the increasing evidence that co-colonization

might be associated with higher colonization densities. Brugger et al (Brugger et al.,

2010) showed that colonization density was significantly higher in nasopharyngeal

swabs containing multiple pneumococcal strains, compared to swabs with a single

strain. Accordingly, Margolis et al (Margolis et al., 2010) have shown, in a neonatal rat

model, that sequential colonization with two pneumococcal strains results in increased

colonization densities to allow the co-existence of the pulsed and the established

strains. This is of great importance due to the increasing interest in using

nasopharyngeal colonization density as a diagnostic tool for pneumonia (Albrich et al.,

2012). Moreover, a study that assessed co-colonization events in samples from healthy

children and children with acute respiratory infection has shown that the latter group

was significantly more co-colonized (Dhoubhadel et al., 2014).

Epidemiology

The epidemiology of co-colonization is generally unknown. There are several

surveillance studies that report colonization by more than one strain in the same host,

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methods of detection and most have not systematically looked at co-colonization (for a

review see (O'Brien & Nohynek, 2003)).

Studies that used the same detection method (serotyping of multiple colonies) suggest

that co-colonization prevalence is highly variable across geographical settings, ranging

between 3% and 36% (Gratten et al., 1989; Hansman & Morris, 1988; Ussery et al.,

1996). Also, there is some evidence that co-colonization prevalence in a given setting

might be associated with the carriage levels in that setting, i.e., settings with higher carriage prevalence will have higher co-colonization levels (Brugger et al., 2010;

Kandasamy et al., 2015; Turner et al., 2011).

There is little data on the effect of vaccination on co-colonization. Brugger et al. (Brugger et al., 2010) have used a highly sensitive method to detect co-colonization in

nasopharyngeal samples collected between 2004 and 2009, spanning PCV7

introduction in Switzerland. They concluded that the rate of co-colonization was similar

before and after the introduction of PCV7 and that it was associated with younger age,

as children younger than 2 years of age were significantly more co-colonized. This

study has also shown that co-colonization rates are independent of the vaccination

status of the child, which is in disagreement with the results obtained in Portugal,

where it was shown that, in the vaccine era, children with four PCV7 doses were

significantly less co-colonized than non-vaccinated children (see chapter II).

The quest for the perfect detection method

The first reports on co-colonization were obtained by successive intraperitoneal saliva

injections in mice to detect multiple serotypes (Gundel et al. (Gundel M, 1933), reviewed in (Shak et al., 2013)). Using this strategy, Gundel et al. reported a 73% rate of co-colonization. Although impractical, this methodology was the basis for the

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by serotyping of several colonies, to detect co-colonization. The latter strategy was

used for several years and constitutes the basis of most reports on co-colonization

(Barker et al., 1989; Gratten et al., 1989; Hansman & Morris, 1988; Huebner et al.,

2000; Ussery et al., 1996). Despite being the most straightforward approach for

detection of co-colonization, serotyping of multiple colonies has proved to be of little

value due to its low sensitivity to detect less abundant serotypes in a nasopharyngeal

sample. Huebner at al. (Huebner et al., 2000) have shown that, to have a 95% chance

of detecting a serotype present in a sample in a relative abundance of 1%, one would

need to serotype 299 colonies. This is highly demanding in feasibility and cost.

For the reasons mentioned above, several alternative serotyping methods have been

developed, taking into consideration the ability to detect co-colonization. The criteria to

define a good detection method rely on the ability of that method to (i) detect minor

serotypes with high specificity, (ii) detect serotypes directly from the nasopharyngeal

specimen, (iii) detect all known serotypes, (iv) distinguish pneumococci from closely

related species, (v) be quantitative, and (vi) be affordable (Satzke et al., 2012; Satzke

et al., 2014).

These methods can be divided into phenotypic and genotypic and some are variants of

the same technology (Table I).

Most phenotypic methods are either bead-based (Park et al., 2000; Sheppard et al.,

2011; Whaley et al., 2010) or blot-based immunoassays (Bogaert et al., 2004c;

Bronsdon et al., 2004). The blot-based methods have the advantage of being

cost-effective because they use highly diluted typing sera but they are technically

demanding and can be of difficult interpretation due to cross-reactions. The

bead-based methods are technically demanding and of difficult implementation, as they

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Turner et al. (Turner et al., 2011) have also developed a phenotypic method based on

latex agglutination. This method uses an enrichment step of the nasopharyngeal

specimen according to the WHO recommendations and resuspension of the primary

selective growth on a saline solution to be tested against all available typing sera,

allowing the detection of virtually all capsular types or groups. The authors were able to

detect minor serotypes that corresponded to down to 25% of the total pneumococcal

population.

The genotypic methods include PCR-based methods, including real-time PCR, and

microarray-based methods. Their main advantage is their higher sensitivity, when

compared to phenotypic methods, which enables the detection minor serotypes

present in a very low abundance that could be below the culturable limit.

Several multiplex-PCR systems were developed that are able to detect co-colonization,

although all of them require a previous enrichment of the nasopharyngeal specimen

(Morais et al., 2007; Rivera-Olivero et al., 2009; Yu et al., 2011). These methods have

the limitation of having low discriminative power in closely-related capsular types, of not

being quantitative and the fact that most of them target a limited number of capsular

types. They present high sensitivity and are of straightforward implementation and

execution.

Real-time PCR based methods have also been developed (Azzari et al., 2010; Pimenta

et al., 2012). These methods are extremely sensitive but are limited by the low discriminative power in closely-related capsular types and by the optimization

difficulties for multiplexing. Azzari et al. (Azzari et al., 2010) have shown, however, the

main advantage of this approach, by demonstrating its ability to detect co-colonization

directly from the nasopharyngeal sample with a four times higher sensitivity than

sequential multiplex PCR. Another key advantage of this method is the fact that it is

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(46)

(47)

The main drawback of both conventional and real-time PCR based methods is the

possible occurrence of false positive results associated with the fact that these

methods use as target fragments of capsular genes that can potentially be present in

other inhabitants of the nasopharynx, such as closely related Streptococcus spp. (Carvalho Mda et al., 2013).

Brugger et al. (Brugger et al., 2009) have developed a PCR-based method which takes

advantage of a highly conserved and specific genetic region within the pneumococcal

species, adjacent to the pneumolysin gene (ply), which encodes for a major virulence

factor and is highly specific for pneumococcus. This variable noncoding region adjacent

to the ply gene (plyNCR) is amplified by PCR and the PCR product is then digested for

restriction fragment length polymorphisms (RFLP). The restriction is performed with

four enzymes used independently and the samples are analyzed by capillary

electrophoresis for exact size assessment of the digestion fragments. When the sum of

the restriction fragments exceeds the size of the undigested product (c.a. 1400 bp),

there is evidence that more than one pneumococcal strain exist in the sample. This

method has high throughput but requires expensive equipment and reagents and a

fairly high level of technical expertise, as interpretation of results can be misguided by

incomplete digestion or presence of closely-related Streptococcus spp. The greatest disadvantage of this method is related to the fact that it does not discriminate the

serotypes of the co-colonizing strains. For this reason, it has to be coupled with another

genotypic method that has this ability.

Genotypic methods based on comparative genomic hybridization (CGH) have also

been developed (Hinds et al., 2010; Tomita et al., 2011; Turner et al., 2011; Wang et

al., 2007). However, to our best knowledge, only the microarray developed by Hinds et al. (Hinds et al., 2010; Turner et al., 2011) has the ability to detect all capsular types described up to now. In this method each capsular gene is targeted by ten 60-mer

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capsular types. As detection is based on fluorescently labeled oligomers, this

microarray is able to quantify the relative abundance of each capsular type in the total

of pneumococcal DNA present in the samples. Despite being very sensitive, this

method is not suitable for detection directly from the nasopharyngeal specimen,

although optimization is ongoing (Satzke et al., 2012). The main drawback of this

method is, however, the requirement of very expensive reagents and equipment and

high level of technical expertise.

Due to the existence of all these different approaches and to the increasing relevance

attributed to co-colonization in vaccine surveillance studies, an initiative was

undertaken under the scope of the PneuCarriage project in which 20 methods were

compared for their ability to detect co-colonization with a good cost/benefit ratio. This

assessment is being done by using a reference collection of spiked samples mixed with

real samples, in which methods with the best performance in detecting minor serotypes

with good positive predictive value will be selected for a second round that consists in

the analysis of 200 field samples (Satzke et al., 2012). First reports announced that

four methods were selected for the second stage of evaluation: the microarray

developed by Hinds et al. (Hinds et al., 2010; Turner et al., 2011), the latex agglutination method developed by Turner et al. (Turner et al., 2011), the plyNCR-RFLP coupled with multiplex PCR, developed by Brugger et al. (Brugger et al., 2009), and the multiplex PCR and real-time PCR developed by Azzari et al. (Azzari et al., 2010).

Co-colonization determinants

Co-colonization has been known to occur for many years, although the factors

determining its occurrence and the interactions that occur between strains inside the

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Several theoretical models have been used to predict which are the determinants of

co-colonization and strain interactions, all of them considering the capsular type as the

decisive factor to distinguish between strains (Auranen et al., 2009; Cobey & Lipsitch,

2012; Zhang et al., 2004). These studies have resulted in the formulation of important

premises about between-strain competition and carriage of multiple serotypes:

co-colonization does not seem to accelerate clearance (Auranen et al., 2009), established

colonization with one serotype reduces risk of acquisition of a second serotype

(Auranen et al., 2009), serotype-specific immunity stabilizes competition, and acquired

immunity to non-capsular antigens reduces fitness differences between strains, being

relevant for competition (Cobey & Lipsitch, 2012; Zhang et al., 2004). Notwithstanding,

these theoretical principles have not been sufficiently addressed experimentally.

Experimental and epidemiological data on this subject is scarce and the bacterial

factors that might affect this phenomenon are generally unknown. In the following

sections information regarding some bacterial factors that might have an impact on

co-colonization and that are relevant in the context of this thesis will be presented.

The contributions of the capsule and genetic background to

(co)colonization

The ability of a strain to colonize an individual is intrinsically related to its ability to resist

clearance. With the objective of determining the invasive potential of different

serotypes, Sleeman et al. (Sleeman et al., 2006) have shown that less invasive serotypes are carried for longer periods. Most studies that have addressed which

factors contribute to the a success of a strain in colonization have focused on the role

of the capsule, as it is the main target of the immune system in the clearance process

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capsule prevents clearance and aggregation, affects colonization and adherence, helps

the pneumococcus to survive in the lungs and spread to the bloodstream, and

contributes to antibiotic tolerance. Pneumococci regulate the amount of capsular

material produced during colonization and invasion. Transparent capsules (thin) are

favored during initial colonization stages to promote adherence to host epithelial cells,

while opaque capsules (thick) are favored during invasion to resist

complement-mediated opsonophagocytosis (Fernebro et al., 2004; Lysenko et al., 2005; Mac &

Kraus, 1950; Morona et al., 2004; Nelson et al., 2007; Weiser et al., 1994).

Weinberger et al. (Weinberger et al., 2009) have shown that the structure of the capsular polysaccharide can predict serotype prevalence. These authors and others

have shown that serotypes with capsule structures with more carbon in their

polysaccharide repeat unit required more energy for capsule production, displaying

thinner capsules. Therefore, these serotypes were less resistant to phagocytosis and

clearance, which correlated with their lower prevalence (Hathaway et al., 2012;

Weinberger et al., 2009). Other capsule-related properties have been studied to explain

the influence of the capsular type on pneumococcal colonization, such as the surface

charge. Li et al. (Li et al., 2013) have shown that the surface charge correlates with the

prevalence of serotypes in colonization, as more negatively charged capsular types are

more prevalent than less charged capsular type and capsules more negatively charged

were shown to repel host immune cells that contribute to clearance, such as

neutrophils and macrophages.

Several epidemiological studies have shown that there is high variability in strains of

the same serotype (Brueggemann et al., 2003; Hanage et al., 2005; Sá-Leão et al.,

2011; Sandgren et al., 2004) and this observation is supported by the increasing

available genomic data (Chewapreecha et al., 2014; Hakenbeck et al., 2001; Hiller et